1. Field of the Invention
This invention relates to diagnostically useful agents, a process for their preparation and their use in diagnostic methods. More particularly, the invention relates to blood-derived delta hepatitis antigen and to its use in the determination and detection of anti-delta antibodies.
2. Description of the Prior Art
The hepatitis agent named delta (.delta.) was first detected by Rizzetto, M. et al using the technique of immunofluorescence in the liver of some patients chronically infected with the hepatitis B virus or with chronic hepatitis B surface antigenaemia (Gut, 1977 18 997-1003). The delta agent (now called hepatitis D virus) has been shown to be associated with, but not part of, the hepatitis B virus. It is hypothesized that the delta agent is dependent on hepatitis B virus for replication because it has not been reported so far in the absence of markers of hepatitis B virus. It has been held that the agent has a putative genome and that it cannot replicate without the help of hepatitis B virus (Rizzetto, M. et al The Journal of Infectious Diseases Vol. 141, No. 5 May 1980). This hypothesis therefore includes the possibility that constituents of the hepatitis B virus or its associated particles and tubules may also be constituent parts of the delta agent. Evidence in support of this hypothesis includes the finding that antibody to the hepatitis B surface antigen (HBsAg) agglutinates particles found in delta-rich cesium chloride density-gradient fractions (Rizzetto, M. et al Proc. Natl. Acad. Sci. U.S.A. Vol. 77, No. 10 pp. 6124-6128, October 1980). Such particles were not agglutinated by antibody to the delta agent.
Evidence that the delta agent is a virus or virus-like entity is based on transmission experiments to chimpanzees and the demonstration of low molecular-weight RNA in semipurified delta enriched cesium-chloride density gradient fractions (Rizzetto, M. et al Proc. Natl. Acad. Sci. U.S.A supra).
It is likely that the delta agent particles have a structure which can be depicted schematically as follows: ##STR1##
The delta antigen is considered to be a protein and appears to have a molecular weight of 68,000.
It is postulated that delta antigen exists in the blood as part of the particle depicted above and as free delta antigen, some of which may be bound to plasma proteins. The infection that is attributed to the delta agent is commonly associated with persons who illicitly abuse drugs by injection. Delta infection is also reported in persons who receive quantities of blood-derived material, and examples of such persons are haemophiliacs.
Since 1980 delta infection has become very common in Ireland among persons who illicitly abuse drugs by injection (Shattock, A. G. et al Irish Journal of Medical Science, Vol. 151, No. 11, 334-338, 1982). Delta infection appears to be almost worldwide and is even found in isolated South American tribes.
Delta infection is reported to be particularly common in Italy even in those not using illicit drugs or receiving blood-derived products. However, such persons carry or are infected with hepatitis B virus or carry hepatitis B markers. Many of these persons are reported to have chronic forms of hepatitis and although it has not been proved, the implication is that the delta agent may be contributory in some way to the acquisition or maintenance of this chronic hepatitis.
Even more alarming are reports of a higher incidence of delta markers in persons with severe or fulminant forms of hepatitis compared with those with benign hepatitis (Smedile, A. et al The Lancet, October 30, 1982 pp. 945-947 and Raimondo, G. et al Brit. Med. J. 286 p845, March, 1983). This has not been proved but the implication is that delta infection is causative of or contributory to the severe and/or fulminant form of hepatitis. It will be appreciated, however, that the whole picture is complicated by the presence of hepatitis B virus.
The Italian subjects referred to above were deemed to have the delta infection because either they were found to have a marker called the delta antigen in their liver by immunofluorescence, or because they were found to have antibody to the delta antigen (anti-delta) in their blood by radio-immunoassay or enzyme-immunoassay techniques. In the latter studies the delta antigen was derived from liver of a patient with chronic delta infection by extraction of hepatocyte nuclei (disrupted by sonication) with 6M guanidine hydrochloride followed by dialysis and boiling of the concentrated dialysate (Rizzetto, M. et al 1979 The Lancet ii 986-990). By any standards the latter extraction method is severe and may result in antigenic alteration.
German Patent Publication DE-A-3116882 relates to a stable form of the delta antigen of Hepatitis B virus without interfering material and associated liver tissue, a method of isolating the delta antigen from liver and its use as a diagnostic agent for detecting corresponding antibodies indicative of associated disease. It is stated (page 7) that "At present, the human liver which contains delta antigen is the only practical source of the antigen".
A paper by Redeker A. G. in April 1983 (Annals of Internal Medicine Volume 98 No.4) shows that to date serum has not been considered a practical source of delta antigen, since detection of delta antigen serum has only been shown in chimpanzee transmission studies and in a few patients in whom delta antigenaemia has been transiently recorded early in acute delta hepatitis. By transient is meant a few days.
The presence of delta antigen in blood of chimpanzees experimentally infected with delta antigen has been demonstrated after treatment of the serum with various concentrations of the detergent known as Nonidet P40, a non-ionic detergent (Rizzetto, M. et al, 1980; Proc. Natl, Acad. Sci. U.S.A. supra). Delta antigen activity was found to be optimal at a final concentration of 0.3%. Since detection of delta antigen required detergent treatment Rizzetto et al deduced that the delta antigen was an internal component of a discrete subpopulation of HBsAg particles.
Because of this apparently rare and transient presence of the delta antigen in the blood of infected persons, blood has not heretofore been considered as a source of antigen in a technique for the detection of anti-delta antibodies.
Delta antigen is considered to be highly immunogenic since antibody thereto appears very rapidly in the plasma of infected subjects. Since one obtains a negative result for delta antigen with anti-delta antibody within a few days of the first appearance of delta antigen, it has been assumed that delta antigen has disappeared. However, it is now postulated by the Applicants that, in fact, the delta antigen may persist in the bloodstream but bound to delta antibody.
To date, the detection of anti-delta antibodies in blood has been carried out with delta antigen derived from human post-mortem liver. The techniques used for the extraction of the delta antigen from liver are not routine to many laboratories and in any case suitable human postmortem liver is very difficult to obtain and potentially hazardous to the surgeon and handlers who risk infection during the extraction process. As a result, few laboratories have been able to carry out research on the incidence and significance of delta infection simply because they lack a laboratory technique for the detection of delta antigen and anti-delta antibodies.
Liver derived delta antigen is in short supply and, therefore, few laboratories have access to suitable reagents for monitoring delta infection. A new source of delta antigen as a test reagent is, therefore, urgently required.
It is an object of the present invention to provide a method of `unmasking` delta antigen or delta antigens (hereinafter referred to collectively as delta antigen) present in blood so that it becomes readily available for use as a diagnostic agent in the detection and determination of different classes of antibodies to the delta agent, including the IgM class of antibodies which are indicative of current infection.